Abstract Melanoma is a tumor of the skin that frequently metastasizes and causes many deaths annually. Although there are effective therapies for melanoma such as checkpoint blockade or BRAF inhibitors, resistance mechanisms are frequently activated. Immunotherapy has become a frontline treatment for metastatic melanoma, but only 20-30% of patients have long term benefit from the treatment. Resistance correlates to the number of CD8 positive T cells that have infiltrated the tumor. Epigenetic regulators that are mutated in melanoma are associated with drug resistance to checkpoint blockade. Understanding the pathways regulated by these chromatin factors will allow for better therapies. We have developed a rapid melanoma model using zebrafish and can model many of the mutations associated with human melanoma. We have created stable transgenic fish with the CD8a promoter driving fluorescent markers and generated primary melanomas in this line to live image the T cells as they enter the tumor. Using this approach, we have found distinct behaviors of T cells that interact directly with melanoma cells. Slow migrating T cells appear exhausted at the borders of tumors, whereas active faster moving T cells interact with the melanoma cells. Based on human melanoma genetics defined by our clinical colleagues, we plan to quantify this behavior in melanomas with mutations in epigenetic regulators such as ARID2 and G9a. Preliminary data shows that G9a inhibition suppresses the enhanced tumor initiation by ARID2 deficiency. Other regulators will be investigated based on human tumor genetics. The effects of these epigenetic regulators on T cell migration into the melanoma will be assessed by live imaging using two photon microscopy. We will study chromatin accessibility of these tumors using ATAC-seq and correlate the results with human tumor accessibility as defined by our group. The correlation of live imaging with chromatin accessibility will help define the mechanisms of resistance of these transcription factors and will provide preclinical information about G9a inhibitors on ARID2 deficient tumors. We will create models with mutations in a variety of transcription factors and epigenetic regulators, particularly those of interest to the other investigators on this grant. Enhancer reporters will also be developed to examine target genes in the tumors and T cells. We also will study the chromatin effects driven by BRAF treatment, focusing on the mechanism of resistance. ETV1, a gene that is amplified in 8% of melanoma, is reorganized on chromatin to activate a network of genes that drive resistance. ETV1 is known to be phosphorylated by MAPK and we plan to interrogate this mechanism using proteomics. By screening an epigenetic chemical library, we hope to reverse the resistance network for therapeutic purposes. We will also investigate the role of ETV1 in the recruitment of T cells to tumors. Our studies, greatly strengthened by our collaborations with Dr. Fisher (G9a and mouse melanoma models), Dr. Liu (epigenetic analyses), and Drs. Wucherpfennig and Rodig (immunological mechanisms and cell profiling) have great impact on the basic mechanism of resistance and will lead to new therapies for the treatment of drug resistant melanoma.